Browsing by Subject "Master's Programme in Translational Medicine"

Sort by: Order: Results:

Now showing items 1-20 of 48
  • Adhikari, Sadiksha (Helsingin yliopisto, 2020)
    Structural variants comprise a large number of variations occurring in the human genome and are detected in many diseases including cancers. To a limited extent, whole exome sequencing (WES) is capable of detecting structural variations (SVs) using algorithms and tools utilizing local assembly, split-reads, discordant read-pairs and read depth methods. However, due to the significantly large size of SVs compared to the reads produced and the presence of repetitive regions in the genome, identification of SVs presents a major challenge. 10X Genomics has developed a technology that requires very low amounts of DNA and uses a linked-reads approach to produce long reads. Recently, linked-read technology has shown promising results in resolving complex SVs. In this thesis, we aimed to assess whether linked-read exome sequencing is able to infer more comprehensive information in SVs compared to WES in multiple myeloma (MM). The disease model was chosen based on the presence of high numbers of SVs in MM patient tumor cells. Here, we report that linked-read sequencing has led to the identification of a potential novel translocation t(1; 14) that significantly impacts the change in expression of genes and could potentially have impact on the prognosis and treatment of multiple myeloma patients. By Long Ranger analysis we detected t(1;14) in six out of eight samples. Further, to study whether the translocation differentially affects the expression levels of any genes, differential gene expression was performed between t(1;14) positive versus t(1;14) wild type samples. The analysis resulted in 107 differentially expressed genes where 4 upregulated and 103 downregulated genes were found in the translocation positive samples. Among the downregulated genes, we found S100A8 and S100A9 genes which are previously shown to be associated with chemoresistance to PAD (bortezomib, doxorubicin and dexamethasone) therapy. The related breakpoints of the event were identified by Manta tool (SV caller) using both linked-read and WES. Therefore, linked-read information does not appear necessary to detect this event. In this study, we found that linked-read sequencing has certain advantages over WES such as low input DNA, increased number and quality of calls and breakpoint information. However, linked-read sequencing technique is limited to the detection of certain SV types in addition to increased cost of sequencing. These two factors must be considered before choosing linked-read sequencing over WES. Somatic mutations and clinically relevant SV were detected equally efficiently by both techniques.
  • Nguyen, Ngoc Anh (Helsingin yliopisto, 2019)
    Immunophenotyping by flow cytometry (FC) is an established practice to identify immune cells and their cellular changes at the single-cell level. Since preserving the structural integrity of cellular epitopes is vital for immunophenotyping, samples should be processed shortly after being collected. However, the requirements of complex facilities and trained personnel for flow cytometry make it challenging to handle samples immediately. Fixation and cryopreservation extend sample shelf life and allow analysing longitudinal samples simultaneously while minimizing technical variation. Nevertheless, usage of whole blood cryopreservation in flow cytometry is limited due to challenges in preserving epitope structures during fixation and detecting dim antigens. This thesis investigates the performances of four commercial whole blood cryopreserving kits; 1) Cytodelics, 2) Stable-Lyse V2 and Stable-Store V2 (SLSS-V2), 3) Proteomic stabiliser (PROT-1), and 4) Transfix. Peripheral blood samples were processed with these stabilising buffers immediately after the collection and cryopreserved until further analysis by flow cytometry. Here, we measured the stability of major immune lineages, T cell subpopulations, and activated neutrophil profiles in samples treated with these commercial whole blood stabilisers. Our flow cytometry data showed that PROT-1, Transfix and Cytodelics maintained the distribution of major leukocyte subsets – granulocytes, T cells, natural killer cells and B cells, comparable to unpreserved samples despite the attenuation of fluorescence intensities. Moreover, these three stabilisers also preserved phenotypes of activated neutrophils upon stimulation with N-Formylmethionyl-leucyl-phenylalanine and Lipopolysaccharides. The upregulation of adhesion molecules (CD11b), Fc receptors (CD16) and granule proteins (CD66b) as well as the shedding of surface L-selectin (CD62L) on activated neutrophils was conserved most efficiently in PROT-1, followed by Cytodelics. On the other hand, none of the stabilisers provided a reliable detection of CCR7 for accurate quantification of T cell subpopulations. COVID-19 is caused by a highly transmissible and pathogenic coronavirus, so-called severe acute respiratory syndrome coronavirus 2 (SARS-COV-2). To test the potential of whole blood cryopreservation kits for flow cytometry in COVID-19 research, we studied the detectability of major leukocyte lineages and granulocyte subsets in longitudinal patient samples processed with Cytodelics. High dimensional analysis with Uniform Manifold Approximation and Projection (UMAP) and Self-Organising Maps (FlowSOM) clustering revealed remarkable stability of CD3, CD15, and CD14 expression in samples stored with Cytodelics. It allowed the detection of lymphopenia and emergency granulopoiesis often found during the acute phase of severe SARS-COV-2 infection. Nonetheless, we could not determine signatures of granulocyte subsets, notably suppressive neutrophils, during the acute and convalescent phases of COVID-19. Variable detection of lowly expressed markers and diminished fluorescence intensities in Cytodelics - preserved samples might have hindered the analysis. In conclusion, this study demonstrates that PROT-1, Transfix, and Cytodelics enabled reliable detection of highly expressed leukocyte markers, whereas SLSS-V2 preservation resulted in the most inaccurate identification of studied markers. Notably, our results show that Cytodelics can be applied in COVID-19 studies to immunophenotype major immune lineages by flow cytometry. Nevertheless, more optimisation is needed for less abundant or fixation-sensitive epitopes to enhance the efficacy of whole blood cryopreservation for flow cytometry.
  • Valkonen, Konsta Valentin (Helsingin yliopisto, 2021)
    Amyotrophic lateral sclerosis (ALS) is the most common adult-onset motoneuron disease. ALS is characterized by a progressive loss of upper and lower motoneurons, resulting in muscle atrophy, paralysis and ultimately in death. Approximately 30,000 people die of ALS annually. There is no cure for ALS, and only two drugs - riluzole and edavarone - have been approved for the treatment of the disease. The complex pathology of ALS contributes to the lack of effective treatments. Several cellular pathologies have been suggested to contribute to the pathogenesis, including ER stress, disruption of calcium homeostasis, oxidative stress and excitotoxicity. Here we describe the cytoprotective effects of C-terminal fragments of the novel proteins with neurotrophic factor properties MANF (mesencephalic astrocyte-derived neurotrophic factor) and CDNF (cerebral dopamine neurotrophic factor) on a toxin model of ALS in vitro. Unlike the classical neurotrophic factors, MANF and CDNF are predominantly localized to the endoplasmic reticulum (ER) and have been shown to alleviate ER stress by keeping the unfolded protein response (UPR) transducers inactive. ER stress is a major component in many neurodegenerative diseases, including ALS, and is a promising therapeutic target for MANF and CDNF. However, the potential of these proteins in ALS treatment remains to be insufficiently described. We used differentiated motoneuron-like NSC-34 cells treated with a range of toxins, modelling different cellular pathologies linked to ALS. After the toxin addition, we treated the cells with MANF and CDNF variants and riluzole and measured the cell viability. The toxin panel consists of tunicamycin, ionomycin and staurosporine. Tunicamycin causes cell death by activating proapoptotic branches of the UPR. Ionomycin is an ionophore and depletes the ER of calcium, thus inducing both UPR-dependent and UPR-independent apoptosis. Less is known about the mechanisms of staurosporine, but it has been shown to induce caspase-3-dependent apoptosis, increase intracellular calcium levels and cause oxidative stress. We hypothesized that both MANF and CDNF variants protect the cells against UPR-dependent apoptosis but not against UPR-independent cell death. We show that MANF and CDNF variants protect the cells against apoptosis induced by tunicamycin, ionomycin and staurosporine. Interestingly, the protein variants mediated the highest protection against ionomycin-induced stress, and they exhibited mild protective effects against staurosporine as well. These findings suggest that MANF and CDNF variants might have a role in maintaining intracellular calcium homeostasis. However, it is possible that staurosporine induces ER stress as well, which would explain the protection conferred by the protein variant. We report that the CDNF variant mediates higher protection at lower concentrations compared to the MANF variant in every toxin assay, whereas the MANF variant mediates higher protection at the highest tested concentration compared to the CDNF variant. We also show that the CDNF variant-mediated protection against staurosporine-induced stress peaked at lower concentrations, and the highest concentration provided distinctively lower, yet significant effect. These data lead us to hypothesize that the protein variants may have a slightly different mode of action, and that they might provide an additive effect when administered simultaneously. We tested a combination of MANF and CDNF variants in cells treated with tunicamycin, ionomycin and staurosporine. However, the combination treatment did not increase the viability more than MANF and CDNF variants independently did. The results answered our questions as well as raised new ones. In the future, the putative calcium-regulating effects of the protein variants should be investigated. The UPR-modifying effects of the drug candidates and toxins need to be assessed by quantifying changes in the UPR marker mRNA and protein expression levels. If it is revealed that the variants have a different mode of action, the possible additive protective effects must be assessed. Finally, a wider toxin panel is needed to fully explore the potential of MANF and CDNF variants in ALS treatment. This study demonstrates the potential of MANF and CDNF variants in protecting motoneurons against several pathological pathways contributing to ALS pathology. However, the mechanisms of action of the variants need further investigation to fully understood their therapeutic potential.
  • Fan, Qiuyu (Helsingin yliopisto, 2020)
    Alzheimer’s disease (AD) is a neurodegenerative brain disorder in which the disease process may take decades until the symptoms become evident. To date, no ideal biomarker has emerged that would enable early detection of AD. Environmental and lifestyle factors are thought to affect the risk of developing AD, possibly through epigenetic mechanisms such as DNA methylation (DNAm). DNAm has been shown to differ in the blood and brain of subjects with AD compared with subjects without AD, suggesting that DNAm may be involved in the pathogenic process of AD. This study aims to detect the difference in blood DNAm at baseline between cases who later developed AD and controls who remained AD diagnosis-free during follow-up in a sample selected from a Finnish population-based cohort. Leucocyte genome-wide DNAm was profiled on approximately 850,000 CpG sites by using Infinium MethylationEPIC assay. Each CpG was regressed on the outcome of AD diagnosis during follow-up, controlling for subjects’ age at sampling, sex, smoking status, blood cell counts, working stress level, slide, and array. Specific differentially methylated positions (DMPs) were further explored using pathway analysis. Finally, the methylation level of the candidate gene (APOE) selected from the literature was compared with the sample of this study. After correction for multiple testing, the later diagnosis of AD was not significantly (adjusted p-value < 0.05) associated with methylation level at the baseline at any DNAm site. There was, however, a robust hypomethylation of DMPs among the cases, as 90.9% of the DMPs (p-value < 0.05) were hypomethylated in the case group. The 200 genes annotated by DMPs with the smallest p-values were involved in two neuronal pathways: “Axon guidance associated with semaphorins Homo sapiens” (p-value = 0.0058, adjusted p-value = 0.065) in Panther 2016 and “Semaphorin interactions Homo sapiens” (p-value = 0.00005, adjusted p-value = 0.078) in Reactome 2016. No significant difference existed in DNAm of the candidate gene (APOE) between cases and controls, while cg26190885 at the promoter region of APOE showed nominal significance (p-value = 0.04). In conclusion, no strong evidence was found to support the hypothesis that systemic changes in DNAm are involved in the pathogenesis of AD or that DNAm marks could be detected in blood before the symptoms become evident. A genome-wide pattern of hypomethylation measured by the Infinium MethylationEPIC assay was observed in the case group, serving as a venue for further investigations.
  • Jalkanen, Nelli (Helsingin yliopisto, 2020)
    Mitochondrial aminoacyl tRNA-synthetases (mt-aaRS) catalyse the charging of tRNAs with their cognate amino acids in mitochondria. Mutations in mt-aaRS cause tissue-specific mitochondrial diseases, especially affecting tissues with high energy expenditure like the nervous system, heart, and kidneys. However, disease mechanisms for the heterogeneous group of diseases have not yet been fully elucidated. Harnessing CRISPR-Cas9 genome editing in induced pluripotent stem cells (iPSC) provides an opportunity to model mt-aaRS mutations in vitro and investigate the effects of individual mutations on cellular phenotype. SARS2 encodes mitochondrial seryl tRNA-synthetase, and its c.1347 G>A mutation causes severe childhood-onset progressive spastic paresis. Here, CRISPR-Cas9 ribonucleoprotein (RNP) complex and associated donor template were used to induce homology directed repair (HDR) the genome of iPSC and knock-in the patient mutation. Guide RNAs were designed and tested for efficiency before electroporation into wild type iPSC. Clonal cell lines were made by low-density seeding and manual colony picking. The expression of pluripotency markers was measured by RT-qPCR. RT-qPCR and Western blot measured SARS2 mRNA expression and protein level respectively. The success and precision of genome editing were analysed by Sanger sequencing, comparing the performance of the different guide RNAs, and screening regions of potential off-target genome editing. Two genome-edited iPSC lines with the SARS2 c.1347 G>A mutation were successfully generated to model the patient mutation. The iPSC lines expressed pluripotency markers and contained no off-target genome editing and modelled the patient’s decrease in SARS2 protein level and mRNA expression. More evidence of differentiation ability is needed before differentiation into the affected cell type (motor neurons) and further disease modelling. The efficiency of CRISPR-Cas9 for genome editing, especially harnessing HDR in iPSC, is an area of future research.
  • Patpatia, Sheetal (Helsingin yliopisto, 2020)
    Antibiotic resistance of pathogenic bacteria has increased in recent years. When antibiotics do not work, alternative therapies are developed to prevent major bacterial epidemics. Phage therapy is one of the alternative possibilities to cure infections caused by antibiotic resistant bacteria. Due to the narrow host range of phages, hundreds or even thousands of phages are required to cover the various bacterial pathogens. For a reliable selection process, high-throughput rapid host range screening of phages is needed to cover the future demands. In addition, collaboration between laboratories is highly important, as the collections of phages of individual laboratories are not broad enough. Thus, the transportation of phages between laboratories is one of the key elements to provide successful phage therapy for patients. The aim of the study was to use gel-based products as protective matrix in phage host-range screening and transportation. The optimal conditions were selected to set a baseline for high-throughput rapid host range screening process, and to set up a ready-to-screen plate assay for phage transportation. In addition, the purpose of the study was to evaluate whether hydrogels could be used as a long-term storage matrix for phages and future product development. Fourteen Escherichia coli phages were used to optimize the liquid culture assay for the E.coli strains. The hydrogel based assays were conducted with two Escherichia and two Staphylococcus phages. For long-term storage, phages were mixed with different consistencies of hydrogels and stored in three different conditions for up to six months at +4oC. The transportation experiments were conducted with phages stored with optimized hydrogel consistencies. The phage viability was measured using liquid culture method. Results show that liquid culture method on microtiter plate is a convenient way to screen bacteriophages in high-throughput assay and that phages can be stored reliably in hydrogel format. When stored in microcentrifuge tubes, phage stability was shown to last for at least six months. When stored as drops on microtiter plate, the phages retained their viability for up to two months. These plates can be used as a robust means for phage transportation.
  • MacKeith, Ada (Helsingin yliopisto, 2019)
    Sleep difficulties have been on the rise for the past decade. Insomnia and sleep difficulties have associations with an increased risk of overall mortality, as well as with a diverse array of complex diseases, such as coronary heart disease, major depressive disorder, fibromyalgia and Alzheimer’s disease. Epigenomics provides information on how environmental factors influence the genome via epigenetic mechanisms, such as DNA methylation. Thus far, epigenome-wide association studies looking at the effects of sleep disturbances on the methylome have provided evidence of distinctive methylation patterns in insufficient sleep, involving biological processes related to neuroplasticity and neurodegeneration. However, more knowledge is needed to determine how the severity of sleeping difficulties influence the methylome. This thesis investigates the effects of increasing sleep difficulties on DNA methylation with an epigenome-wide association study. The study sample is derived from the Health 2000 general population survey. Subjects were divided into three different groups by their self-reported level of sleeping difficulty, and methylation measurements performed from whole blood samples utilizing the Illumina Infinium MethylationEPIC kit, encompassing >850,000 CpG sites. To identify differentially methylated sites, a multivariable regression model was used with age, gender, smoking, alcohol use, cell type distribution and plate and array data as covariates. None of the differentially methylated CpG sites identified remained significant after multiple testing correction. To gain more information regarding which biological processes the methylated sites may be part of, those CpG sites with an uncorrected p-value of <0.0005 were subjected to pathway analysis. Notable significant pathways included oxytocin- and serotonin receptor-mediated signalling pathways and Alzheimer’s disease-amyloid secretase pathway. Altogether, six pathways remained significant after multiple testing correction, with a total of 12 different genes appearing in them. Furthermore, a post-hoc regression analysis was conducted between these 12 genes and their corresponding CpG sites, and health-related quality of life questionnaire responses. Significant results included associations between sleep, and discomfort and symptoms (including pain). As an additional analysis, a database search was conducted to learn more about the genes’ functionality at the level of phenotype. Results included some variant trait associations to sleep, Alzheimer’s disease and cognitive performance. The associations to Alzheimer’s disease and cognitive performance warrant further research with a similar additive model, perhaps with a larger sample.
  • Ahveninen, Lotta (Helsingin yliopisto, 2022)
    Objectives. Ageing is accompanied by neurobiological changes, such as changes in grey matter (GM) volume and cortical thickness, that mediate a gradual cognitive decline, which can, in turn, be potentially offset by stimulating leisure activities. Choir singing is an especially feasible musical activity with positive effects on physiological, psychological, cognitive, and social functioning in old age. Research investigating the effects of choir singing on the ageing brain is limited. As part of the Brain, Ageing, and Vocal Expression (BRAVE) project, this study aimed to investigate the effect of ageing and choir singing on GM structure. Methods. Using a cross-sectional design and voxel-based morphometry (VBM) and surface-based morphometry (SBM), this study compared GM structure between young (20-39 years; n=35), middle-aged (40-59 years; n=34), and old (60-90 years; n=31) participants and investigated the interaction of age and choir singing on GM structure with amateur choir singer (n=54) and controls (n=46). Results and conclusions. Age had a significant and widespread effect on GM structure, with old participants showing lower GM volume and cortical thickness than young (in bilateral sensorimotor, auditory/language, visual, and limbic areas, midbrain, and cerebellum) and middle-aged (in right visual cortex, thalamus, hippocampus and left auditory cortex) participants. Middle-aged participants also showed lower GM volume and cortical thickness than young participants (in bilateral sensorimotor, language, and visual areas, basal ganglia, cerebellum, and right hippocampus and amygdala). These results corroborate the current understanding of neurobiological ageing. No significant interaction of age and choir singing was found on GM structure, which could be explained by methodological factors. Further research is needed to determine whether choir singing can support brain structure or function across healthy ageing.
  • Sinkko, Matilda (Helsingin yliopisto, 2021)
    In this master’s thesis, in vitro neuromuscular junction (NMJ) model was set up using microfluidic devices. Additionally, the effect of R878H/R878H mutation in MCM3AP gene that causes an early-onset peripheral neuropathy on NMJ formation and maintenance was studied. To study human NMJs that significantly differ from other mammal NMJs is challenging and new models to study the function of these complex and highly specialized structures are needed. Induced pluripotent stem cells (iPSC) and motor neurons were characterized with gene expression studies using qRT-PCR and with immunocytochemistry studies using commonly known markers for pluripotency and motor neurons. NMJs were studied in 2D co-cultures and with microfluidic devices. Gene expression studies were conducted from 2D co-cultures and co-cultures in microfluidic devices provided detailed information of the localization and morphology of NMJs. Expression of essential genes for NMJ formation together with immunocytochemistry results with alpha-bungarotoxin (BTX) staining showed that NMJs were formed in both control and R878H/R878H mutant cell line co-cultures. There was a trend of lower gene expression levels of NMJ essential genes in the R878H/R878H mutant line compared to the control line and also immunocytochemistry results indicated impairment in NMJ formation in the mutant line, but further studies are needed to validate the effect of R878H/R878H mutation on the NMJ formation. In future, functional studies could be conducted to investigate whether these NMJs are functional and the information from the motor neuron terminal is conveyed to the muscle membrane.
  • Their, Anna (Helsingin yliopisto, 2021)
    The contact site between the endoplasmic reticulum and mitochondria, also known as the mitochondria endoplasmic reticulum contact sites (MERCS), have a crucial role in maintaining the homeostasis within the cell. Across the MERCS multiple functions, such as regulation of calcium (Ca2+) homeostasis, lipid metabolism, ER stress, mitochondrial quality control (MQC) and regulation of unfolded protein response (UPR) take place. These processes have been shown to be implicated in numerous different neurodegenerative diseases, such as Parkinson’s disease. Parkinson’s disease is the second most common neurodegenerative disease that at the moment has no cure. The main obstacle in developing a neuroprotective treatment for the disease is the limited understanding of the key molecular events leading to neurodegeneration. One of the things in Parkinson’s disease that has eluded scientists for years is the selective death of the dopaminergic (DA) neurons in substantia nigra pars compacta. One hypothesis that could explain the selective death is the Ca2+ hypothesis, looking at the Ca2+ vulnerability of SNpc DA neurons as a plausible cause leading to the selective cell death. This project focused looking at the protein level and morphological changes of the ER and MERCS in stressed neurons, hypothesizing these as possible sites that contribute to the neuron vulnerability, as they are known to be the key modulators of the intracellular Ca2+ homeostasis. This study looked closer at two MERC proteins, GRP75 and BAP31, and one ER protein, SERCA2, to see how they are affected in stressed dopamine-like neurons. Firstly, the in vitro model was established by differentiating SH-SY5Y neuroblastoma cells to dopamine-like neurons expressing tyrosine hydroxylase. Three different molecular compounds were tested as possible stressors affecting the Ca2+ homeostasis within the neurons, and we concluded that thapsigargin, a commonly used stressor to model PD like pathology, leads to the highest measurable ER Ca2+ depletion. Lastly, we quantitatively and qualitatively analyzed the effect of 24-hour treatment with each stressor on the differentiated SH-SY5Y neurons. Thapsigargin treatment lead to an increased level of GRP75 and SERCA2. A slight increase in BAP31 was also detected after thapsigargin treatment, but no apparent changes of the ER morphology were detected. The results, together with previous research, show GRP75 to be a possible contributor to the pathology of the disease, but further research is needed to see if it could be a possible target for treatment.
  • Kaaja, Ilse (Helsingin yliopisto, 2020)
    Bone marrow failure (BMF) is a condition where the bone marrow fails to produce enough functional blood cells leading to peripheral blood cytopenias. Inherited BMF is often a consequence of germline mutations in DNA repair pathway, telomere maintenance, or ribosome biogenesis -related genes and results in up to 20-40% risk of developing a hematological malignancy. Recently, biallelic germline mutations in the gene ERCC6L2 have been identified to cause inherited BMF leading to the accumulation of somatic TP53 mutations and acute myeloid leukemia (AML M6) with dire prognosis. ERCC6L2 is a DNA repair protein that has also been indicated in mitochondrial function. The aim of this thesis was to study the ERCC6L2 protein expression and cellular metabolism in ERCC6L2-derived BMF. The metabolic profile in ERCC6L2-derived BMF was studied in patient-derived fibroblasts using a Seahorse XFe96 Analyzer. The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured at multiple time steps when cells were in standard cell culture (10mM) glucose concentration or low (1mM) glucose concentration. The protein expression was studied in fibroblasts and peripheral blood mononuclear cells (PBMCs) with immunofluorescence assay and Western blotting. The results of this thesis demonstrate a differential metabolic profile in the patient-derived cells. In normal glucose, they thrive exhibiting a higher basal OCR, ATP-related respiration, mitochondrial reserve capacity, and maximal respiratory capacity compared to the control. Contrarily, in low glucose the patient-derived cells struggle and show a lower basal OCR, ATP-related respiration, reserve capacity, and maximal respiratory capacity than the control implying decreased substrate availability in the mitochondrial respiratory chain or mitochondrial dysfunction. Immunofluorescence assay suggests that ERCC6L2 is expressed in the patient-derived cells supporting truncating mutations observed in RNA sequencing. In order to improve the treatment and clinical outcomes in inherited BMF, understanding the role of altered mitochondrial metabolism in ERCC6L2-derived BMF and its progression to AML M6 calls for further studies.
  • Rinne, Nea (Helsingin yliopisto, 2022)
    Aims: Reading ability is a fundamental skill in the modern society, yet some individuals have difficulties in learn-ing to read and write. There is a lot of variability in reading skills, and one reason that can cause reading difficulty is a neurodevelopmental disorder called dyslexia. It is the most common learning disability, and the core deficit in dyslexia lies in word decoding, which is the process of connecting letter combinations into their corresponding auditory representations. Dyslexia is familial and is recognized to have strong genetic background. A dozen dyslexia susceptibility genes have been suggested, but DYX1C1, DCDC2 and KIAA0319 have been associated with dyslexia most commonly. The function of these genes is however not yet fully understood. In previous studies variation in these genes have been linked to struc-tural brain alterations in left hemispheric regions where language is mostly processed. The aim of this study was to examine the connection between dyslexia susceptibility genes DCDC2, DYX1C1 and KI-AA0319 and variation in brain activity during reading tasks in the left middle temporal gyrus (MTG), infe-rior Frontal Gyrus (IFG) and intraparietal sulcus (IPS), by combining functional magnetic resonance imag-ing data and genetic data in a neurotypical population. Previous studies have reported that weaker reading skills are associated with decreased brain activity in these regions, and reading incongruent sentences has been associated with increased brain activity in the left IFG and MTG. Methods: During fMRI, participants were presented with sentences with illogical and logical endings, and judged them as either congruent or incongruent, in distracted and undistracted conditions. Auditory speech stimuli were used as distractor. Regions of Interest analyses were conducted to examine brain activation in the aforementioned brain regions during distracted and non-distracted reading separately for different allelic groups in single nucleotide polymorphisms of the three genes. Results and Conclusions: DYX1C1 showed significant interaction with brain activation in the IPS. A significant interaction of DCDC2 with logic was found in the IFG and IPS showing that individuals carrying susceptibility alleles have reduced brain activation when reading incongruent sentences. Additionally, DCDC2 showed inter-action with distraction in the IFG, as individuals carrying susceptibility alleles had reduced brain activa-tion when a speech distractor was presented. In the MTG, there was a significant interaction of DCDC2 with logic and distractor showing that in different allelic groups, speech distractor modulated the activa-tion elicited by incongruent sentences in different ways. These results provide a link between variation in dyslexia susceptibility genes and brain activation during reading. Previous studies have mostly linked dyslexia susceptibility genes to structural brain alterations, and dyslexia and lower reading skills have been linked to variation in brain activity. The current study therefore expands the current understanding of genetic basis on reading and linguistic processing.
  • Jagdeo, Joanna (Helsingin yliopisto, 2020)
    Merkel cell carcinoma (MCC) is a rare, neuroendocrine carcinoma of the skin that is known to have poor prognosis. It is associated with the Merkel cell polyomavirus (MCPyV) and majority of cases harbor this infection. Other risk factors include older age, the male sex, Caucasian skin and increased ultraviolet exposure. Increased lymphocyte invasion into the MCC tumor microenvironment has been reported to infer better survival, but better mechanisms understanding why this occurs this is needed. CCL3 is a chemokine that is implicated in a variety of inflammatory conditions like viral infections and exhibits pro-inflammatory activity mainly through its chemoattractant abilities. In cancer specifically, it functions within the tumor microenvironment by encouraging the trafficking of leukocytes to the tumor site. Transcriptomic data of CCL3 was studied in a cohort of 102 Finnish MCC patients to observe its association with survival, and a variety of clinical-pathological features. The presence of CCL3 in cells was later investigated via immunohistochemistry in 30 formalin-fixed paraffin-embedded Finnish MCC primary tumor tissue samples with varying mRNA expression of CCL3. Macrophages and lymphocytes were found to stain positively for CCL3 and were found exclusively in tumor surroundings. CCL3 was also found to exhibit a MCC-specific survival benefit in patients that harbored higher expression (p=0.031), and was found to be associated with MCPyV positivity (p=0.032). These preliminary findings help establish CCL3’s role in the immune response against MCC and support the need for further studies looking at CCL3 both as a prognostic marker and potential adjuvant therapeutic.
  • Nowlan, Kirsten Helene Anna-Marie (Helsingin yliopisto, 2020)
    Mucosal associated invariant T (MAIT) cells are a fairly recently described population of innate-like T cells. In humans, MAIT cells represent an exceptionally abundant population in the blood, where they account for 1–10% of all T cells. However, compared to conventional T cells, which can display an almost unlimited T cell receptor (TCR) repertoire, the specificities of MAIT cell TCRs are limited. This evolutionarily conserved subset displays a semi-invariant TCR which recognises riboflavin metabolites, produced by a wide range of bacteria and fungi, and presented on the major histocompatibility complex (MHC)-class I related (MR1) molecule. The function and significance of MAIT cells in health and disease have only started to be unravelled, and it is becoming increasingly clear that MAIT cells are also modulated in non-microbial diseases. Interestingly, MAIT cells have been shown to exhibit a relatively high expression of the transcription factor, Helios, compared to most other T cell subsets. The function of this translational activator and repressor, encoded by the gene IKZF2, in the MAIT cell population remains obscure. This study focused on the functional role Helios may play in regulating the activation of MAIT cells. Thus, by using siRNA to silence Helios expression, and flow cytometry to analyse any potential alterations in MAIT cell activation markers, we aimed to be able to characterise the kinetics and functional role of Helios in peripheral MAIT cells of healthy individuals. Here, we clearly established a striking upregulation of Helios in MAIT cells following 24 hours of stimulation. Moreover, we were able to achieve a >50% knockdown of Helios at the protein level, in this subset of T cells. Nevertheless, no significant difference in any of the activation markers we investigated was present between the MAIT cells with reduced Helios expression and their controls. This could, however, of occurred as a result of the toxicity that the transfection had on the functionally of the T cells. From these results, it is difficult to conclude any clear role for Helios in the activation of MAIT cells, and consequently, further research needs to be performed before any clear conclusions can be drawn.
  • Arffman, Maare (Helsingin yliopisto, 2021)
    Uterine leiomyomas are common smooth muscle tumours, with a prevalence as high as 80%. Even though they are benign, they present severe symptoms such as heavy menstrual bleeding, pelvic pain and reproductive dysfunction. Uterine leiomyomas can be classified to conventional tumours and leiomyoma variants based on their histopathology. The tumours usually harbour one of the three driver alterations: MED12 mutations, HMGA2 overexpression or biallelic FH inactivation. Known risk factors for leiomyoma development are African ancestry, family history and age. Uterine leiomyomas are most typically treated by surgery, through either uterus preserving myomectomy or by definitive hysterectomy. This Master’s thesis is continuation of a study from Äyräväinen et al. 2020, a retrospective study of 234 patients undergoing myomectomy at Helsinki University Hospital during 2009-2014. The aim of this study was to analyse how many of these patients had developed recurrent leiomyomas and how often the tumours in subsequent operations were potentially clonally related. In addition, clinical characteristics associated with the operations were analysed. In total 18% of these patients had recurrent operations, leading to the screening of 77 individual uterine leiomyomas from 32 patients. The mutational statuses were studied systematically with molecular screening using Sanger sequencing and immunohistochemistry. Altogether 33 tumours from 21 patients were found to have identical mutational status with a tumour from the original study. Of these tumours, 14 had a MED12 mutation. All the MED12 mutations were found in exon two affecting either codons 44 or 36. Six tumours had HMGA2 overexpression, and eight tumours were FH deficient. Five tumours were triple negative for all studied alterations. Whereas 81% of the patients had had two removal operations, the rest of them had had three to five operations. The years between operations ranged from performing them on the same year to performing them ten years apart. Even though most of the recurrent tumours were sporadic, almost half (43%) of them had identical mutations, suggesting that though uterine leiomyomas usually arise independently, some might be clonally related. The mutational distribution was different in the recurrent tumours than in uterine leiomyomas in general, indicating that in addition to germline predisposition, the driver related characteristics might also contribute to the potential of recurrence and to the likelihood of developing clonal lesions. Tumours harbouring MED12 abnormalities were the least probable to be clonally related. The tumours showing identical HMGA2 overexpression were likely clonally related. The number of identical FH deficient ULs was high, but not unexpected, since all the patients harbouring the mutation in the recurrent tumours had HLRCC, and therefore having a predisposition. Most surprisingly, all patients with recurrent triple negative tumours had identical mutation statuses in the recurrent tumours, which points to previously unknown clonal development of these lesions. Most of the patients with more than two surgeries had recurrent mutations, suggesting that multiple surgeries might indicate the development of clonally related tumours. However, further research is required to confirm the clonal relationships and to investigate the pathological nature of the tumours with different driver alterations.
  • Bouhlal, Jonas Otto Vilhelm (Helsingin yliopisto, 2022)
    Despite of great advancements in the field of cancer therapy in the past decades, the 5-year survival of acute myeloid leukaemia (AML) patients remains low with high mortality especially in elderly patients, in whom the disease is most often observed. Poor prognosis often results from complex heterogenous molecular abnormalities defining the progress of the disease, while making it more difficult to treat due to intensive treatments only being feasible for younger patients. Our increased understanding of cancer immunology and the potential of immunotherapy has, however, led to promising therapeutic innovations, which give hope for discovering long-lasting and effective treatment options. Natural killer (NK) cell-based immunotherapies are amongst the emerging novel therapeutic approaches that aim to target malignant cells with less toxicity and improved applicability. Using high-throughput drug sensitivity and resistance testing combined with single cell RNA (scRNA) sequencing, this study focused on finding drug compounds that could synergise with NK cells to improve their effectiveness in killing leukemic cells. In this study, many drugs showed promising results in being able to potentiate NK cell cytotoxicity, with daporinad and pevonedistat showing the most notable differences when compared to controls. The potentiating effect of Janus kinase (JAK) inhibitors also suggested a method of increasing NK cell activity against leukemic cells through downregulation of major histocompatibility complex (MHC) class I molecules. In conclusion, findings shed light on the synergetic potential of drugs and NK cells, giving hope for clinically relevant findings following further validation and testing.
  • Pällijeff, Pieti (Helsingin yliopisto, 2021)
    Recently, several novel post-translational modifications (PTMs) have been identified as important regulators in biology. Succinylation, the reversible addition of a succinyl group from a free succinyl-CoA into a protein lysine, is one such novel PTM. The last decade of research has unveiled succinylation as a powerful regulator of metabolism, prevalent in every organism it has been studied in and with functional effects on target proteins in several key metabolic pathways. A major contribution of this thesis is to catalogue the recent advances in succinylation research into the most comprehensive literary review currently available on succinylation. While the biological role of this PTM is being established, the relevance of succinylation in human disease has remained unclear. Meanwhile, mitochondrial DNA depletion syndrome caused by defective SUCLA2 (SUCLA2 disease) is a progressive hereditary mitochondrial disease with no available treatment. SUCLA2 disease is caused by defective mutations in the ß-subunit SUCLA2 of the TCA cycle enzyme succinyl-CoA synthetase. While the characteristic manifestations, including impairment of respiratory complexes, and the etiological mutations in this disease are well established, the pathogenic model for SUCLA2 disease has remained incomplete. As succinyl-CoA synthetase shares a substrate, succinyl-CoA, with succinylation, this thesis set out to probe SUCLA2 mutants for a potential succinylation phenotype. An extensive hypersuccinylation phenotype was characterized in fibroblasts and tissue samples from SUCLA2 mutant patients by immunochemical methods. The hypersuccinylation target identities in SUCLA2 mutants were revealed with proteomics by mass-spectrometry. Hypersuccinylation in SUCLA2 mutants was shown to be enriched in proteins participating in mitochondrial energy metabolism, including respiratory complex proteins. In addition, several novel metabolic phenotypes were characterized in SUCLA2 mutants with metabolomics by mass-spectrometry, most prominently a significant depletion of aspartate metabolism. While identification of extensive hypersuccinylation in SUCLA2 mutants establishes a novel concept of succinylation relevance in human metabolic disease, the prospect of altered regulation of the respiratory complexes due to hypersuccinylation lays the foundation for a novel pathogenic model for SUCLA2 disease. Meanwhile, the observed novel metabolic phenotypes significantly contribute to the current understanding on SUCLA2 mutant metabolism and inspire a hypothetical model on how the defective succinyl-CoA synthetase could be circumvented in the TCA cycle of SUCLA2 mutants.
  • Siskovs, Klims (Helsingin yliopisto, 2021)
    STK11/LKB1 is a tumor suppressor gene and mutated in 18% of lung adenocarcinomas. Tumor suppressor liver kinase B1 (LKB1) is known to activate adenosine monophosphate-activated protein kinase (AMPK) and 12 AMPK-related kinases (ARKs) by phosphorylating a conserved threonine residue in their T-loop region. A number of studies focused on investigating the influence of LKB1-AMPK signaling on cancer cell proliferation. However, there is no systematic study for identifying the critical LKB1 kinase substrates in suppressing lung cancer cell growth. In this project, the LKB1-deficient lung adenocarcinoma cell line A549 cells were sequentially overexpressed with constitutively active mutants of AMPKα1, AMPKα2, MARK1, MARK2, MARK3, MARK4, NUAK1, NUAK2, SIK1, SIK2, SIK3. The overexpression status was confirmed at both genetic and protein levels by qPCR and Western blotting, correspondingly. In vitro growth assays demonstrated up to 33% reduced growth rate of A549 cells overexpressing AMPKα1, AMPKα2 and NUAK1. Furthermore, siRNA knockdown of the selected substrates in LKB1-overexpressing A549 cells significantly rescued the cell growth defect. These findings suggest, that AMPKα1, AMPKα2 and NUAK1 kinases are critical for LKB1-mediated cell growth defect in lung adenocarcinoma.
  • Tripathi, Shivanshi (Helsingin yliopisto, 2020)
    Multiple Myeloma (MM) is the second most common hematologic malignancy. Despite the advancements in treatment approaches in the last decade, the prevalence of refractory disease leading to relapsed cases has been a major challenge. A wide range of intricate genetic heterogeneity demonstrated by myeloma patients is a credible explanation for the diverse treatment responses observed in patients sharing the same treatment regimens. Pertaining to this, the study aims to identify predictive gene expression biomarkers that forecast response to BCL2 inhibitor venetoclax and treatment outcome to proteasome inhibitor bortezomib. In this study, samples from MM patients were characterized into sensitive and resistant, (1) based on ex vivo response to venetoclax treatment (Resistant n=21; Sensitive n=21), and (2) based on their bortezomib treatment outcome in clinical profiles (Resistant n=12; Sensitive n=15). Associations between the different gene expressions and drug responses were studied using statistical and bioinformatic tools. As a result, we identified that significant (p-value <0.05) overexpression of 36 genes and downregulation of 38 genes appeared to confer resistance to venetoclax drug response in MM patients. Additionally, the functional association of these genes with pathways was determined using a pathway enrichment tool. Furthermore, the study provided evidence that cytogenetic alterations t(11;14) and t(4;14) are significantly (p-value <0.05) associated with differing venetoclax response in MM patients. These findings demonstrated that gene expression biomarkers and chromosomal translocations play a significant role in regulating venetoclax drug response in MM, which can be further utilized to personalize treatments for patients. The knowledge obtained from this work best applies in personalized medicine; whereby fitting treatments to an individual patient’s genomic landscape will enhance patient outcome.
  • Stadelmann, Christian (Helsingin yliopisto, 2019)
    Induced pluripotent stem cells (iPSCs) can be derived from somatic cells by transgenically expressing the four transcription factors OCT4, SOX2, KLF4, and C-MYC. This technology has revolutionised the stem cell field, yet cellular reprogramming is still inefficient and slow. To become fully applicable in regenerative medicine, the robust generation of safe and high-quality iPSCs from patient samples is essential. Various methods and potent reprogramming factors have been described to date. Yet, none have been able to circumvent these limitations markedly. The recently published activator-mediated approach (CRISPRa) is considered to be more physiological compared to the forced transgenic expression as the cell’s own genes are activated. Here, guide RNAs (gRNAs) mediate sequence-specific recruitment of non-cutting Cas9 (dCas9) activator proteins to the promoter region. Unlike other methods, it holds great multiplexing capacity and can also target enhancer and non-coding sequences. CRISPRa reprogramming still needs to be optimised since its efficiency is low. Thus, we aimed at enhancing this aspect and the temporal kinetics by targeting the micro RNA (miRNA) clusters 302/367 and miR-371-373, which both have been described as powerful cell fate regulators. We demonstrate successful reprogramming by targeting the miR-302/367 promoter alongside OCT4, SOX2, KLF4, C-MYC, LIN28A, REX1, NANOG, and EEA-motifs with CRISPRa. Activating the miRNA cluster results in a 2.5 fold efficiency increase in human foreskin fibroblast (HFF) reprogramming compared to the published basal CRISPRa system, quantified by staining for alkaline phosphatase. In HFFs, the CRISPRa efficiency is now comparable to the commonly used transgenic approach. Aiming to clarify the molecular mechanisms of these results, we characterised the expression of direct and downstream targets of miR-302/367 at different time points throughout the reprogramming process. Furthermore, validated with immunocytochemical stainings, the generated bona fide iPSCs express pluripotency markers and spontaneously differentiate into the three germ-layers, both signs of high-quality iPSCs. Beyond that, we report that miR-302/367 activation appears to result in earlier iPSC colony formation resulting in faster proliferating stem cell colonies shown with live-cell imaging. Employing a conditionally stabilised activator construct, we further show that with miR-302/367 targeting, the dCas9 activator expression seems to be required for only a short time period, sufficient to induce pluripotency. At the end of the project, the miR-302/367 cluster targeting was optimised and the best-working gRNAs were selected for further studies, which when combined further increase the CRISPRa-induced expression of the miR-302/367 cluster markedly. All in all, this study demonstrates that non-coding genetic elements like the miR-302/367 cluster can be targeted with CRISPRa, and its targeting significantly improves the reprogramming efficiency. Implications of the study for regenerative medicine and future steps are discussed.